A study is proposed which will yield specific information about the mechanism by which Ca 2 ion is actively transported and released by the sarcoplasmic reticulum from skeletal and cardiac muscle. The study will make use of highly sensitive fluorescent probe and indicator methods together with the stopped-flow rapid kinetic method, in conjunction with an experimental design taking into consideration the current membrane-biophysical concepts of allosteric mechanisms, cation-ligand interactions, protein-lipid interactions, membrane potential and mechanisms for its generation, and membrane surface potential. The Ca 2 ion pump protein of the SR, the Ca-Mg-ATPase will be characterized in situ with respect to its high affinity Ca 2 ion binding, the kinetics (Km and Vm) of its passive transport reaction, the kinetics (Km and Vm) of its active transport reaction, the inhibition of these parameters by other cations and the dependence of these parameters on the phospholipid composition. Experiments will be carried out to determine the maximal gradient which the Ca 2 ion pump can establish and how this is affected by the ion concentrations in the medium. A variety of methods will be employed to determine whether the Ca 2 ion pump operates in an electrogenic fashion or by a Ca 2 ion for Mg 2 ion or K ion exchange diffusion mechanism. Fluorescent probe methods will be employed to determine whether membrane potential is involved in the active accumulation or triggered release mechanisms. A fluorescent probe method will be applied to determine whether surface charges and changes in surface potential are involved in the Ca 2 ion sequestration after the active transport step. These data will be related to a detailed model for Ca 2 ion pump function and Ca 2 ion binding inside the sarcoplasmic reticulum, and will be compared to data on maximal steady-state uptake. Skeletal and cardiac sarcoplasmic reticulum will be compared with respect to the above parameters.